A sensitive and specific two stage polymerase chain reaction (PCR) method was developed for the simultaneous amplification and detection of specific genomic sequences of hepatitis B virus (HBV) and hepatitis C virus (HCV) in donor sera. Initially, HCV-RNA was reverse transcribed to cDNA. This cDNA, and DNA from HBV, were then co-amplified by using primer pairs derived from conserved regions of HBV and HCV nucleotide sequences. The specificity of PCR products was confirmed by liquid hybridization analysis using 32P-end labeled oligomer probes specific for HBV and HCV nucleotide sequences. Independent sets of sera, positive and negative by PCR for either HBV-DNA or HCV-RNA, were used as controls. Nine donor sera, antibody reactive for both HBV (HBsAg and anti-HBc) and HCV (anti-HCV), were tested. Our assay detected HBV and HCV-specific genomic sequences in 9/9 sera reactive for both HBV and HCV antibodies. The ability to co-amplify and detect HBV and HCV sequences, non-isotopically may be particularly useful in the clinical laboratory setting.